This invention relates to carbazine dyes and derivatives thereof for purposes of pH measurement. More particularly, the invention relates to carbazine dyes, compositions containing carbazine dyes bonded to solid supports, and methods of using such carbazine dyes and compositions for measuring pH.
Hydrogen ion concentration or pH is an extremely important parameter in biological and many chemical systems. Many chemical and biological reactions require close regulation of pH for reactions to occur properly. For example, a complex natural process for the control of pH occurs in human blood, which normally has a pH of about 7.4. Variations of even a few tenths of a pH unit can cause serious illness or death. The carbon dioxide concentration of the blood affects the pH significantly because of the propensity of CO.sub.2 to combine with water to form carbonic acid. Hemoglobin plays a crucial role in regulation of blood pH by transporting carbon dioxide from the capillaries to the lungs and also by playing a role, with plasma proteins, as a buffer. The lungs ordinarily remove carbon dioxide from the blood as fast as it is formed, thus helping to maintain a constant pH. The kidneys also have a primary role in regulating the hydrogen ion concentration of the intracellular and extracellular fluids by secreting acidic or basic constituents when these deviate from normal and restoring the balance thereof.
Although a variety of techniques have been developed to measure pH, they generally are based on either electrochemical or optical principles. A standard laboratory pH meter, for example, comprises a standard electrode of known potential, a special glass electrode that changes potential depending on the concentration of hydrogen ions in the solution into which it is dipped, and a potentiometer that measures the potential between the two electrodes. The potentiometer reading is automatically converted electronically to a direct reading of the pH of the solution being tested. Indicators, on the other hand, are dyes that change optical properties, such as absorbance or fluorescence, with changes in pH. The greatest sensitivity of indicators to small changes in pH occurs when the equilibrium constant between the acidic and basic forms of the indicator, i.e. the pK.sub.a, is near the pH of the medium being measured.
As a broad generalization, optical pH measurement is considered inferior to electrochemical techniques, primarily because factors other than hydrogen ion concentration, such as temperature, ionic strength, and protein concentration, affect the dyes and interfere with pH measurement. Nevertheless, optical techniques have strong advantages where cost and size are concerned. Among the optical techniques, methods based on fluorescence are more sensitive than those based on absorbance due to the well known sensitivity advantage for measuring emitted versus absorbed light. Unfortunately, fluorescence emission from typical dyes is substantially more sensitive to interfering factors than is absorbance. Measurement of pH-dependent emission intensity in single cells or on fiber optics with a single excitation wavelength suffer spurious results related to dye concentration, photobleaching of the dye, and cell thickness or path length.
A solution to the problem of dye concentration is to determine the ratio of the amount of fluorescence at a fixed wavelength with excitation at a pH-sensitive wavelength to the amount of fluorescence at the same wavelength with excitation at a relatively pH-insensitive wavelength. This method is commonly used to estimate the pH inside cells with fluorescein derivatives, e.g., Paradiso et al., 325 Nature 477 (1987), and is practical for suspensions of cells and in homogeneous fluids in a research fluorometer or microscope. It is usually impractical, however, to produce two different wavelengths of light of known intensity for exciting fluorescence in flow systems, including flow cytometers and fiber optic systems, for continuous monitoring of pH of flowing fluids, such as blood. U.S. Pat. No. 4,945,171 describes xanthene dyes having a fused (c) benzo ring that exhibit the advantages of being able to measure two emission maxima with excitation at only one wavelength, selectivity in exciting the acid and base forms independently and measuring their emission at either single or dual wavelengths, and measuring characteristic pH-dependent absorption or fluorescence excitation spectra. Compared to the carbazine dyes that are the subject of this invention, these xanthine dyes exhibit lower fluorescence, less stability, greater temperature sensitivity, and smaller Stokes shift, and are difficult to immobilize on a solid support.
R. Hill et al., The Phenol Dyestuff of Liehermann as an Acridan Derivative, J. Chem. Soc. (C) 2462 (1970), describes an acridan derivative, 7-hydroxyspiro [acridine-9,1'-cyclohexa-2', 5'-diene]-2(9H), 4'-dione, that has been used as an oxidation-reduction indicator. This compound and related acridan derivatives, 4',7-dihydroxyspiro [acridine-9,1'-cyclohexane]-2(9H)-one; 7-hydroxy-2',3',5',6'-tetramethylspiro[acridine-9,1'-cyclohexa-2', 5'-diene]-2(9H), 4'-dione; 9,9-diphenyl-7-hydroxyacridin-2 (9H)-one; and 9,9-dimethyl-7-hydroxyacridin-2(9H)-one, yield blue solutions in sulfuric acid which turn red on dilution, this color being due to protonation of the free base. The neutral forms of the compounds are yellow in most solvents. A method of synthesizing these compounds is also disclosed.
In view of the foregoing, it will be appreciated that pH-sensitive dyes and methods of use for determining pH, with reduced sensitivity to potentially interfering factors and substantially improved pH measurement performance in biological systems, most of which function in the pH range of 5 to 9, would be a significant advancement in the art.